In biological and biomedical research as well as in forensic investigations, very small quantities of biological material have to be isolated at a sample location, transported from the sample location to a depositing location and subsequently analyzed. The samples are frequently taken from laboratory preparations such as tissue sections, which may, for example, rest on specimen slides suitable for microscopy. Especially in forensic examinations, a sample frequently has to be picked up from irregular or opaque surfaces as, e.g., from parts of some piece of clothing or a cigarette butt. Such samples have to be picked up from a definite spot as accurately as possible, i.e. a particular sample must be selected and isolated out of many samples situated at close spaces. This sample must then be taken to a depositing location without getting contaminated; ideally, this process is documented or at least monitored. The samples must be deposited at a depositing location—for example, an Eppendorf vessel or a biochip—as accurately as possible as required by the depositing location or vessel.
There are several approaches in prior art to solving the said problem, none of which is satisfactory. Many pick-up techniques have been developed in connection with microdissection by means of laser beams. Before taking the sample, a laser beam is used to cut out pieces from tissue placed on a specimen slide. Most of the techniques developed for taking up the cut-out tissue are based on the application of layers of adhesive film.
For example, WO 97/13838A1 describes a selectively activable transfer surface: The tissue section on a specimen slide is covered with a transparent transfer film, which itself is attached to a slide. The transfer film is a polymer having thermoplastic properties, i.e. it fuses when heat is applied to it. By means of infrared laser irradiation, the film can be stuck with pinpoint accuracy to the bits of tissue to be isolated, so that after microdissection, when the film together with the slide is lifted off the sample, only these bits of tissue adhere to it. The sample bits cut out can then be deposited in a reaction vessel either with or without the transfer film.
A similar approach is followed in WO98/36261A1 and WO 98/35215A1. Here, a multilayer transfer film is described, which also has thermoplastic properties and contains at least one metal film and a supporting layer. This film is mounted on a carrier, this carrier being shaped as the lid of an Eppendorf vessel. The lid with the film is brought into contact with the sample; laser treatment makes the sample bits of interest adhere to the lid. After the microdissected material has been lifted off, the lid can be placed on the Eppendorf vessel, thus closing the vessel, and the sample can be brought into contact with a reaction solution.
EP 1 250 583 B1 describes a method in which the sample is covered with a foil. The pick-up tool, a carrier with an adhesive coat, is placed on the foil. Subsequently the sample portions of interest are isolated by means of laser microdissection, with the laser severing the tissue and the foil. In this way, only the material cut out adheres to the carrier when the pick-up tool is lifted off the sample surface. Here again, the carrier is preferably shaped as the lid of an Eppendorf vessel; alternatively, a piece of adhesive tape can be used.
Another solution is described in EP 0 879 408 B1. Here again, microdissection is performed at first. By a laser pulse, the microdissected material is then released from the specimen slide and ejected up, where it hits the lid of an Eppendorf vessel. Preferably, the lid has an adhesive coat, so that the material sticks to the lid. Alternatively, an adhesive disc of foil may be used as the capturing surface, which is then placed into an Eppendorf vessel.
The solution approaches described above have several disadvantages, though. Assembly of the carrier layers for the adhesive material is laborious; moreover, the methods necessitate the use of a laser, either for microdissection and simultaneous activation of the adhesive properties of the carrier material, or for subsequent ejection of the dissected material from the specimen slide onto an appropriate carrier. In forensic examinations, for example, the application of such methods is limited, as the sample carriers used there do not readily allow microdissection. Also, ejecting an object from the sample is a step that involves a certain inaccuracy, whereas in forensic examinations, in particular, it must be ensured that actually the desired sample bit has been taken. In addition, as inverse microscopes are used for laser microdissection, the capability of documentation and monitoring is greatly restricted. Exact deposition of the objects at the depositing location intended for them is not guaranteed. In addition, if the samples are very small, e.g. if they are single cells only, the methods described above, in which the isolated material is taken up in an Eppendorf lid, cannot be used, as the volume of the reaction liquid is so small as to make safe detachment of the cell from the lid unlikely, so that, in the subsequent analysis, the necessary chain reaction for amplifying the material cannot be released.
In other methods known in prior art, various tools are used instead of carrier films or the like, for taking up the isolated material.
In WO 97/13838A1, an adhesive tip is described with which bits of tissue can be taken up from specimen slides. The tip is dipped into a mixture of a commercial polyterpene-based resin and xylene. This solution is used as an adhesive. The sample is then taken up with the tip and adheres to the resin-xylene mix. The material taken up is transferred to an analytical vessel. This vessel contains a reaction solution, which cancels the adhesive action of the tip or the resin-xylene mix. The material taken up thus detaches from the tip and remains in the solution. For the next application the tip is again dipped into the adhesive solution. As this is still the same tip, freedom from contamination is not guaranteed.
The procedure described in WO 97/13838 suffers from the same disadvantage. Here, a sharp-edged glass pipette is described, with which bits of tissue can be dug out, which can then be held to the inlet of the capillary by suction. If the suction ceases, the sample can be deposited. It is a disadvantage, though, that especially very small samples—such as single cells—will easily remain stuck to the capillary and fail to detach from it when the suction ceases. Here again, freedom from contamination is not guaranteed.
EP 0 539 888 B1 describes a selecting apparatus for cell clusters and for cells enclosed in gel granules. The apparatus comprises, among other items, a capillary for taking up the objects. This capillary is dipped into a starch-like adhesive, which makes the cell stick to the tip of the capillary. For depositing the cell, the capillary with the cell adhering to it is moved to above a capturing vessel containing a liquid. By means of compressed air blown through the capillary, the adhesive and the cell detach from the tip and drop into the liquid. Apart from the circumstance that the method is suitable only for larger samples, the depositing process cannot be reproduced, so that it is difficult to document, and, moreover, incapable of precisely positioning the cell clusters. In addition, when the cell is blown off, adhesive is blown off with it, which may cover the cell, thus making analysis difficult if not impossible.
DE 198 04 800 A1 describes a solution in which a needle is used as the transfer tool. Picking up and depositing are assisted by suction or pressure, electrostatic or magnetic interactions. WO 97/13838 also describes a needle to which biological material sticks because of electrostatic interaction. An essential disadvantage of using a needle as transfer tool is that the cell, when being released from the needle, cannot be precisely positioned. This disadvantage is detrimental especially if Eppendorf vessels are used, where pin-point accuracy in positioning the object onto the single-drop quantity of reaction liquid is important. If, e.g., the cell is dropped into an Eppendorf vessel by switching off the magnetic action, it is quite possible for the cell to stick to the wall of the vessel rather than getting into contact with the reaction liquid, and thus fail to be analyzed. Also, due to the electrostatic forces, unwanted picking-up of further material cannot be excluded. The probability for this to happen is high especially if—as in forensic examinations—samples are picked up from, e.g., articles of clothing consisting of synthetic fibers, which easily become electrostatically charged.
WO 2005/033668 A1 describes another microdissection method, in which the tissue cut out is fixed to the specimen slide electrostatically. The tissue cut out can then be picked up electrostatically by means of an electrode or sucked to a relatively broad contact surface measuring about 500 μm in diameter and provided with air channels. The air channels have diameters of 8 μm only. The tissue section is then deposited on a sticky substrate. As in case of the needles described above, the transfer of smallest biological objects, such as single cells, by means of an electrode cannot be reproduced. If, on the other hand, the contact surface is used, the large diameter of that surface makes depositing with pin-point accuracy impossible. The large dimension of the contact surface further bears the risk of contamination.
Finally, WO 2004/046734 A1 describes an apparatus for harvesting cells and cell colonies from liquid cultures and semisolid media. A cell is sucked in through a glass capillary. A robot arm then moves the glass capillary to a suitable analysis carrier, e.g. a microtitration plate, where the cell is deposited. Intended for high sample throughputs, the method described in WO 2004/046734 A1 is ill-suited for the investigation of single samples. Also, this method requires a liquid as a taking-up and depositing medium.